It is well known to scientists and physicians that the number and size of cells in a tissue sample is an important factor in determining whether tissue is healthy or diseased. For instance, the number of cells in parts of the brain is a fundamental determinant of behavior and cognitive ability. Even a small deficit or surplus in the number of neurons will have long-lasting effects on performance (e.g., Parkinson's disease, Huntington's chorea and perhaps even schizophrenia).
In the prior art there are numerous suggestions of how to count cells or particles in samples of tissue. One of the more widely used methods was described by Abercrombie in a paper entitled "Estimation of Nuclear Populations From Microtome Sections", Anat. Rec., Vol. 94, pp 239-247 (1946). Using Abercrombie's method, sections of tissue are typically cut in thicknesses of 5 to 20 microns. A counting frame is placed in the eyepiece of a conventional compound microscope and cells in each section are then counted at a magnification of 500 or higher. All cells, cell nuclei or nucleoli that are inside the frame are counted. In addition, those cells that cross 2 out of 4 edges of the four-sided frame--by convention, the right and upper edges--are also counted. In contrast, cells that cross any part of the bottom or left edges of the frame are not counted. The procedure is repeated from the top to the bottom of the section until all cells at all depths have been counted. However, during this procedure no account is actually taken of the three dimensional position of cells, and no attempt is made to determine the number of cells at the top and bottom surfaces of the section that were split or dislodged by the knife during cutting.
A more accurate counting method was described by Howard et al in a paper entitled "Unbiased Estimation Of Particle Density In The Tandem Scanning Reflected Light Microscope", Journal of Microscopy, Vol. 138, Part II, May, 1985, pp 203-212 (1985). Howard et al. suggest the direct optical examination of a cube of tissue. They point out that the sample cube is not physically sectioned but rather that it is examined in an intact state. A particular cube is chosen and the particles or cells that lie entirely inside the cube and half of those which transect its surfaces are counted.
In specific, Howard et al. studied unsectioned semiopaque materials (entire bones) that could not be examined with a normal compound light microscope. Therefore, Howard et al were constrained to use a tandem scanning reflected light microscope (TSRLM), an expensive special-purpose microscope that accepts only directly reflected light. Such an instrument is not designed for use with conventional sectioned tissue. For a description of the TSRLM, see "Tandem-Scanning Reflected Light Microscope" by Petran et al, Journal of the Optical Society of America, Vol. 58, No. 5, May, 1968, pp 661-664.
The method of Howard et al. suffers from several additional drawbacks. It does not take into account the fact that the number of particles or cells can actually change during the preparation of a tissue sample. For instance, if the sample is prepared by microtome cutting, cells/particles can either be sectioned, pushed deeper into the sample, or pulled out of the sample. These effects are illustrated in FIG. 1 by cell 10, cell 12 and pit 14, respectively. If any cube used as suggested by Howard et al. includes such a section of tissue with altered cells, an erroneous count may occur.
In addition, the method of Howard et al. lacks a convenient way to define the cubic counting box. The upper and lower surfaces of the counting box are defined by reference to the microscope's fine-focus scale. The operator must therefore examine the fine-focus control after each movement to see whether the limits of the counting box (cube) have been reached. Thus, after each examination, the operator must glance at the picture, make the particle count and move the focus control, reexamine the position, and repeat the process iteratively until the entire depth dimension of the cube has been traversed.
Accordingly, it is an object of this invention to adapt a modified Howard et al. method to use with a conventional compound light microscope and for examination of typical sectioned materials used in histology, pathology and research.
It is still another object of this invention to facilitate the operator's definition of a counting box so that the method is easier to implement.
It is a further object of this invention to provide an improved apparatus for counting cells in a sample, which avoids inaccuracies introduced by sample preparation.